Tài liệu Báo cáo khoa học: Expression of an a-1,3-glucanase during mycoparasitic interaction of Trichoderma asperellum docx

Expression of an a-1,3-glucanase during mycoparasitic
interaction of Trichoderma asperellum
Luis Sanz
1
, Manuel Montero
2
, Jose
´
Redondo
3
, Antonio Llobell
1
and Enrique Monte
2
1 IBVF-CIC Isla de la Cartuja, CSIC ⁄ Universidad de Sevilla, Spain
2 Centro Hispano Luso de Investigaciones Agrarias, Universidad de Salamanca, Spain
3 Newbiotechnic S.A. (NBT), Seville, Spain
Trichoderma species have been widely used in agricul-
ture as biocontrol agents [1]. This genus has been
extensively studied owing to their ability to rapidly col-
onize substrates [2], induce systemic acquired resistance
in plants [3], their potential for promoting plant
growth [4] and their antagonistic activity against a
wide range of plant pathogenic fungi [5]. The inhibi-
tory effect of their antibiotics [6,7] and cell wall degra-
ding enzymes (CWDEs) [8] against many plant
pathogens is often cited as important aspects of their
antagonistic activity. In addition, the role of Tricho-
derma spp. in the interaction with plants has recently
been reviewed [9]. The increase in knowledge of
Trichoderma has supported the use of these micro-

organisms for biocontrol as whole cells, protein formu-
lations and expressed genes in transgenic plants [10,11].
The highly active nature and diversity of Tricho-
derma enzymatic systems, which include glucanases,
chitinases, proteases, lipases, esterases and DNAses
[8,12] have led to their successful use in environmental
and industrial biodegradation [13], composting [14],
textiles [15], food and feed production [16], and pulp
and paper treatment [17].
a-1,3-Glucanases (EC 3.2.1.59), also named mutan-
ases, are extracellular enzymes able to degrade poly-
mers of glucose bound by a -1,3-glycosidic links.
According to their amino acid sequences, a-1,3-glucan-
ases are grouped inside family 71 of the glycosyl-
hydrolases [18] and are classified as endo-hydrolytic
when two or more residues of glucose are released as
reaction products, and exo-hydrolytic when glucose
monomers are the final reaction products.
The presence of these enzymes has been described in
bacteria, such as Bacillus circulans [19], Flavobacterium
sp. [20], Microbispora rosea [21] and Streptomyces
chartreusis [22]; and filamentous fungi, such as Asper-
gillus nidulans [23] and Penicillium purpurogenum [18].
However, Trichoderma has been the source for the
Keywords
Botrytis cinerea, a-1,3-glucanase;
mycoparasitism; Trichoderma asperellum
Correspondence
E. Monte, Centro Hispano Luso de
Investigaciones Agrarias, Universidad de

Salamanca, Edificio Departamental, Plaza
Doctores de la Reina s ⁄ n., 37007
Salamanca, Spain
Fax: +34 923 224876
Tel: +34 923 294532
E-mail:
(Received 18 October 2004, accepted 18
November 2004
doi:10.1111/j.1742-4658.2004.04491.x
Trichoderma species have been investigated as biological control agents for
over 70 years owing to their ability to antagonize plant pathogenic fungi.
Mycoparasitism, one of the main mechanisms involved in the antagonistic
activity of Trichoderma strains, depends on the secretion of complex mix-
tures of hydrolytic enzymes able to degrade the host cell wall. The antifun-
gal activity of an a-1,3-glucanase (EC 3.2.1.59, enzymes able to degrade
a-1,3-glucans and also named mutanases) has been described in T. harzia-
num and its role in mycoparasitic processes has been suggested. In this
study, we report on the purification, characterization and cloning of an
exo-a-1,3-glucanase, namely AGN13.2, from the antagonistic fungus
T. asperellum T32. Expression at the transcription level in confrontation
assays against the strawberry pathogen Botrytis cinerea strongly supports
the role of AGN13.2 during the antagonistic action of T. asperellum.
Abbreviations
CWDE, cell wall degrading enzyme.
FEBS Journal 272 (2005) 493–499 ª 2004 FEBS 493
purification of a high number of proteins with this
activity. To date, there have been described four pro-
teins with a-1,3-glucanase activity in T. harzianum
[18,24–26] and one in T. reesei [27]. Only two have
been cloned [18,25] and one has been overexpressed

[18]. The comparative study of these two sequences
has demonstrated that both are almost identical pro-
teins from two different Trichoderma strains.
The function performed by these enzymes in the fun-
gal metabolism is not clear, although it may be con-
nected with the morphogenesis of the cell wall [28],
mobilization of a -1,3-glucan from the cell wall in
energy starvation conditions [23,29], or the degrada-
tion of a-1,3-glucan from other fungi during mycopar-
asitic interactions [25].
Applications derived from the use of these enzymes
are related to their antifungal effect against phyto-
pathogenic fungi containing a-1,3-glucan in their cell
wall, like Fusarium oxysporum [30], and to the presence
of its substrate in the dental plaque as one of the main
components, responsible for the accumulation of
microorganisms on tooth surfaces and the consequent
development of caries [31].
In this article, we report on the purification and
molecular characterization of an exo-a-1,3-glucanase
(EC 3.2.1.84), named AGN13.2, and the cloning of the
corresponding gene, named agn13.2, from T. asperel-
lum. We also show that expression of the gene and
enzyme secretion occur when T. asperellum grows
under simulated antagonism. The expression of
agn13.2 during in vivo assays against the strawberry
pathogen Botrytis cinerea strongly supports the role of
AGN13.2 in the antagonistic action of T. asperellum.
Results
Biochemical properties of AGN13.2

Physicochemical and kinetic parameters of AGN13.2
are summarized in Table 1. The release of reducing
sugars was detected only when using mutan as sub-
strate. The main product detected was glucose, sug-
gesting an exolytic mode of action for AGN13.2. Low
levels of cellotriose, three linked glucose residues,
could also be identified after longer incubations, which
may represent the lowest of the substrates that
AGN13.2 could not degrade due to its obliged linear
character.
Protein sequence
The N-terminal peptide of the purified protein was sub-
jected to sequencing and an eight amino acid sequence
was obtained. These residues were: ASSADRLV.
Degenerate primers were designed according to this
sequence and a highly conserved internal sequence pre-
sent in other mutanases (WNDYGES). AGN13.2 pre-
sented an overall protein sequence identity of 79 and
77% to the previously cloned a-1,3-glucanases in
T. harzianum CECT2413 [25] and T. harzianum
CBS243.71 [18], respectively. A multiple sequence align-
ment was carried out with a-1,3-glucanases from
Trichoderma (Fig. 1).
Regulation of agn13.2 and agn13.1 expression
Regulation studies carried out in liquid phases show
that the highest transcript levels for agn13.2 and
agn13.1 were found for B. cinerea cell wall inductions.
However, no agn13.2 and agn13.1 mRNA was detec-
ted in conditions of carbon and nitrogen source star-
vation and chitin induction (Fig. 2). Regulation

studies carried out in the solid phase show that
agn13.2 is induced during the interaction of T. asper-
ellum and B. cinerea, despite the presence of glucose
in the culture media; meanwhile, no signal was detec-
ted in the T. asperellum vs. T. asperellum interaction
(Fig. 3). No signal was detected during the interaction
of T. harzianum and B. cinerea and T. harzianum with
itself.
Table 1. Biochemical characteristics of a-1,3-glucanases in Trichoderma sp.
Origin
T. asperellum
CECT20539
T. harzianum
CECT2413 [25]
T. harzianum
SP234
R
[18]
T. harzianum
CCM-F470 [26]
T. harzianum
QMZ779 [24]
T. reesei
QM6A [27]
M
r
SDS ⁄ PAGE (kDa) 75 75 75 67 – 47
IP-chromatofocusing 6.1 7.5 6.7–7.5 7.1 7.1 –
Inactivation T (°C) 55 50 – 45 – –
Optimum T (°C) 45–55 55 50–55 40 – 50

Optimum pH 5 5 3.5–5 5.5 6 4.5
K
m
(mg mutantÆmL
)1
) 0.8 1.5 – 1.2 – 1.2
Mode of action Exo Exo Exo Exo Exo Endo
N-glycosylation No No No – – –
Mycoparasitic a-1,3-glucanase from T. asperellum L. Sanz et al.
494 FEBS Journal 272 (2005) 493–499 ª 2004 FEBS
Discussion
The essentially pure mutanase had a molecular mass of
 75 kDa after SDS ⁄ PAGE and 132 kDa after gel
filtration chromatography. This result suggests that
AGN13.2 could be a dimeric protein in solution, unlike
another purified a-1,3-glucanase from T. harzianum
CCM-F470, which is probably a tetramer [26]. The kin-
etic constant K
m
for the purified protein was similar and
its specific activity was also in the range reported for
enzymes isolated from T. harzianum CECT2413 [25],
T. reseei QM6A [27] and A. nidulans [29].
Substrate specificity of AGN13.2 was similar to that
reported for the enzyme isolated from T. harzianum
[27], showing a high specific recognition of a-1,3-glu-
cans with a-1,6 branches.
The observed optimum pH and thermostability are in
the range obtained for the T. harzianum CECT2413
mutanase [25] and other enzymes from T. harzianum

CCM-F470 (pH 5.5) [26], T. harzianum QMZ779
Fig. 1. Alignment of T. asperellum AGN13.2 (Accession no. AJ784420) with homologous sequences of T. harzianum (Accession nos.
AAF27911, CAC80439) [18,25]. Identical amino acids in two or more sequences are shaded. The alignment was carried out with
DNASTAR
using MEGALIGN (CLUSTAL) with a gap penalty of 10. The putative mutan-binding region is the one comprised between the two residues
marked with asterisks [18].
L. Sanz et al. Mycoparasitic a-1,3-glucanase from T. asperellum
FEBS Journal 272 (2005) 493–499 ª 2004 FEBS 495
(pH 6) [24], B. circulans (pH 5.5) [19] and Flavobacte-
rium (pH 6.3–6.9) [20]. Thermostability studies suggest
that, as described for other glucanases from Tricho-
derma [32], the binding of the enzyme to its substrate
confers a higher thermal stability to the protein.
Previous regulation studies of the different proteins
characterized with a-1,3-glucanase activity were carried
out in T. harzianum [25] and A. nidulans [23]. These
studies suggested that AGN13.1 from T. harzianum is
an enzyme involved in mycoparasitism, whereas the pro-
tein MutA of A. nidulans allows the mobilization of the
mutan as energy source from its own fungal cell wall.
Regulation studies carried out in liquid phases show
that AGN13.2 and AGN13.1 are induced specifically by
the presence of B. cinerea cell walls. Interestingly, chitin,
a polymer commonly used to establish simulated myco-
parasitic conditions and reported as inducer of several
enzymes related to mycoparasitism [33,34], is not able to
induce the expression of either agn13.2 or agn13.1.
Regulation studies carried out in confrontation assays
in solid phase between B. cinerea and T. asperellum and
T. harzianum, respectively, strongly support the involve-

ment of AGN13.2 and not AGN13.1 in the mycopara-
sitic process under these conditions. The differential
expression of the two AGN13 orthologues in two strains
representing two different biocontrol biotypes of Tricho-
derma (asperellum and harzianum) could be related to
differences in antagonistic behaviour and ⁄ or host range
between these strains and perhaps between the two bio-
types. In connection with this idea, it is worth mention-
ing that both strains display clear distinctive host range
and antagonistic abilities in controlled assays (unpub-
lished results).
Gene expression in the host–Trichoderma interaction
area during in vitro confrontation assays has already
been reported for some other extracellular cell wall
degrading enzymes produced by Trichoderma, such as
endochitinase CHIT42 [35,36]. Interestingly, both
enzymes, AGN13.2 and CHIT42, were purified from
T. asperellum supernatant after mutan affinity purifica-
tion (data not shown). This indicates a common induc-
tion of both antifungal CWDEs in the presence of
fungal cell walls as well as either an association
between the two proteins or a significant affinity of
CHIT42 for mutan.
Experimental procedures
Strains and culture conditions
T. asperellum CECT20539, T. harzianum CECT2413 and
Streptococcus mutans CECT4034 were obtained from the
Spanish Type Culture Collection (CECT, Valencia, Spain).
A
B

Fig. 2. Expression profile of agn13 orthologues in T. asperellum
CECT20539 and T. harzianum CECT2413 under different growing
conditions. RNAs were extracted from mycelia grown from T. asp-
erellum CECT20539 (A) and T. harzianum CECT2413 (B) for 8 h
without a carbon source (1), on 2% glucose pH 5.5 (2), 2% chitin
pH 5.5 (3), 0.5% B. cinerea cell walls pH 5.5 (4), 2% chitin pH 3 (5)
and under nitrogen starvation (6).
AB C
Fig. 3. Expression of agn13 orthologues in T. asperellum CECT20539 and T. harzianum CECT2413 during mycoparasitic interaction. (A) Sche-
matic representation of the confrontation assay, samples were taken from the interaction area (In) between Trichoderma strains (T) and
B. cinerea B98 (Bc) grown in PDA plates. RNAs were extracted from mycelia grown of T. asperellum CECT20539 (B) and T. harzianum
CECT2413 (C) during mycoparasitism simulation in liquid culture using fungal cell walls induction (1), during Trichoderma vs. Botrytis confron-
tation in plate assay (2) and during Trichoderma vs. Trichoderma confrontation in plate assay (3).
Mycoparasitic a-1,3-glucanase from T. asperellum L. Sanz et al.
496 FEBS Journal 272 (2005) 493–499 ª 2004 FEBS
B. cinerea B98 was isolated in our laboratory from infected
strawberries. For protein production a two-step growing
method was used: Trichoderma was grown in Mandel’s
minimum medium [37] supplemented with 2% of glucose
( 10
5
conidiaÆmL medium
)1
) in a rotary shaker
(150 r.p.m.) at 25 °C. After 48 h the mycelium was filtered,
thoroughly washed with 2% magnesium chloride and
water, and transferred to a new flask containing Mandel’s
minimum medium supplemented with different carbon
sources (replacement medium). In the purification of
AGN13.2, 0.5% of B. cinerea cell walls were used as car-

bon source. Mutan, a-1,3-glucan with some a-1,6-glucan
(dextran) side chains, was prepared by growing Streptococ-
cus mutans as described in Wiater et al. [26].
Protein purification and biochemical properties
The purification of AGN13.2 from T. asperellum cultures
was based on ammonium sulfate precipitation of the super-
natant (90% saturation), its affinity towards mutan, chro-
matofocusing and gel filtration as main steps, following the
same procedure and methodology as described in Ait-
Lahsen et al. [25]. The purified AGN13.2 activity was tested
against several polymers with glycosidic linkages using
0.5 mgÆmL
)1
of each substrate: mutan (a-1,3- and a-1,6-glu-
can), nigeran (a-1,3- and a-1,4-glucan), soluble starch
(a-1,4- and a-1,6-glucan), pustulan (b-1,6-glucan), laminarin
(b-1,3-glucan), carboxymethilcelullose (b-1,4-glucan) or chi-
tin (polymer of NAG linked by b-1,4-glycosidic bonds).
Activity on these substrates was measured by reducing
sugars quantification by Somogyi [38] and Nelson [39]
method, except for chitinase activity that was determined as
described in De la Cruz et al. [40]. The products from
hydrolysis by the purified AGN13.2 were applied to a
HPLC Aminex HPX-42A column (Bio-Rad, Barcelona,
Spain); diffraction index of the eluate was used for the
detection of the products. Glucose and cellulose oligosacha-
rides (2–5 polymerization degree) were used as standards.
Substrate controls were considered in each determination.
Thermal stability of the enzyme was determined incubating
the purified protein at temperatures from 25 to 70 °Cin

50 mm sodium acetate for 30 min and then measuring the
remaining enzymatic activity adding mutan. For optimum
pH determination phosphate buffer was used at pH values
between 2 and 3, acetate buffer at pH values between 4 and
5, and phosphate buffer at pH values between 6 and 8. In
all cases the concentration of the buffer was 50 mm.We
used MALDI-TOF MS combined with PNGase F (New
England Biolabs, Herts, UK) treatment to identify its
N-glycan structures and their sites of expression.
Protein sequence and degenerate primed PCR
Amino terminal sequencing from the purified AGN13.2
was carried out at the National Center of Biotechnology
(CNB, Madrid, Spain) following Edman degradation
method. Degenerate primers were designed according to the
sequence obtained and an internal region highly conserved
in other mutanase proteins. These were AGN1 [5¢-GCI
WSIWSIGCIGAYMGIYTIGT-3¢] and AGN2 [5¢-SWYT
CICCRTARTCRTTCCA-3¢], respectively. T. asperellum
chromosomal DNA was used as template under the follow-
ing conditions: 94 °C, 40 s (denaturation); 52 °C, 1 min
(annealing); 72 °C, 2 min (extension); repeated for 40 cycles
and a final extension step of 2 min at 72 °C. We used
100 pmol of each primer in 25 lL reactions.
RNA extraction and RT-PCR
RNA extractions for RT-PCR and northern blotting were
carried out using TRIZOL (Invitrogen, Barcelona, Spain)
following manufacturer’s directions. To obtain the cDNA
sequence encoding AGN13.2, specific primers were designed
according to the previous amplified genomic sequence.
AGN3 [5¢-GCCGTAGTCGTTCCACGTGATAATC-3¢]

was used to clone the 5¢-end of agn13.2 mRNA using
SMART-PCR system (Clontech, Palo Alto, CA USA) and
AGN4 [5¢-GCAGATCGTCTTGTCTTTTG-3¢] was used to
clone the 3¢-end of agn13.2 mRNA using RACE-PCR system
(Roche Diagnostics, Barcelona, Spain). RNA was extracted
from mycelia grown for 8 h with 0.5% B. cinerea cell walls.
Regulation of agn13.2 expression
Regulation of the previous cloned a-1,3-glucanase in
Trichoderma [25] was also considered. Northern blots were
performed using Hybond N+ (Amersham Biosciences,
Barcelona, Spain) membranes and Ultrahyb (Ambion,
Cambridge, UK) as hybridization buffer following manu-
facturer’s instructions. RNA was extracted from mycelia
grown for 8 h in different induction media. During direct
confrontation experiments, agar plugs cut from growing
colonies of B. cinerea were placed in PDA plates covered
with sterile cellophane sheets. Mycelia were allowed to
grow for 48 h and then plugs from growing colonies of
Trichoderma were placed 5 cm away from the B. cinerea
plug. Control plates were inoculated with two Trichoderma
plugs (Trichoderma vs. Trichoderma). Mycelia for RNA
extractions were collected from the interaction area in a
range of 12–24 h after both fungi touched each other.
Equivalent zones were harvested from control plates.
Acknowledgements
This work was supported by the Spanish Ministry of
Science and Technology (MCYT), Fundacio
´
n Ramo
´

n
Areces (Madrid, Spain) and the European Commission
(TRICHOEST project). L. Sanz is a recipient of a FPI
fellowship from MCYT. We thank Dr Samuel Ogueta
L. Sanz et al. Mycoparasitic a-1,3-glucanase from T. asperellum
FEBS Journal 272 (2005) 493–499 ª 2004 FEBS 497
for his help in MALDI-TOF analysis and I. Chamorro
for the technical assistance.
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